自噬介导油性佐剂免疫增强作用的机制研究

Due to the strong and unique effect on immunoenhancement, oily adjuvants have shown a good prospect for vaccine and drug applications; meanwhile, oily adjuvants are used in establishing a variety of autoimmune disease animal models, in which they might play important roles in initiation and activation of the complex immune responses. However, the mechansim of oily adjuvant-induced immunoenhancement remains to be fully understood. Our previous study has found that the oily adjuvants such as pristane could induce the enhancement of autophagy level in macrophages and arthritis model, providing a crucial clue for further pinpointing the molecular mechanism of their immunoenhancement effect. In this project, we will firstly utilize the oily adjuvants including pristane, squalene and mineral oil to stimulate macrophages and dentritic cells, and observe the autophagy changes by detecting the double-membraned structure of autophagosomes under electron microscopy and LC3-I/II convention. And the regulatory role of autophagy in adjuvant-induced immunoenhancement will be verified by autophagy pathway intervention by using specific inhibitors and RNAi of Becn1. Then we will detect the kinase activation of various signaling pathways by using protein phosphorylation microarrays and immunoblotting in normal cells, oily adjuvant stimulated cells, oily adjuvant stimulated cells with autophagy intervention, to screen which signalling pathways mediate autophagy-induced immunoenhancement. And the positive pathway will be confirmed by using specific inhibitors and RNAi. Finally, we will establish the animal models for rheumatoid arthritis and systemic lupus erythematosus by using pristane and squalene, and detect the autophagy levels in immune organs and target organs. In addition, in vivo intervention to autophagy pathway using lenti-virus will be performed to observe the effects of disease progression and the changes of inflammatory pathways. These results would help us clarify the precise molecular mechanism of the immunoenhancement effect of the oily adjuvants, improve the development of novel vaccines and drugs, and provide the novel insights into the pathogenesis of autoimmune diseases.

我们前期发现降植烷等油性佐剂可诱导巨噬细胞及关节炎动物模型自噬增强，但油性佐剂诱导免疫增强的机制仍不清楚。本项目首先采用降植烷、鲨烯、矿物油等油性佐剂刺激巨噬细胞和树突状细胞，通过电镜观察双层膜结构和LC3-I/II转化检测等反映自噬水平，并通过自噬抑制剂和RNAi干预自噬通路，确定其在佐剂诱导免疫增强中的介导作用；然后在正常细胞、佐剂刺激的细胞、佐剂刺激的自噬干预细胞中，通过信号通路的磷酸化抗体芯片和免疫印迹技术筛查关键信号激酶的活化情况，继而通过信号通路抑制剂/RNAi干预确定自噬介导免疫增强的通路；最后构建降植烷、鲨烯诱导的类风湿性关节炎、系统性红斑狼疮等疾病动物模型，检测免疫器官和靶器官自噬水平，并采用慢病毒在体干预自噬，观察对疾病及相应炎性通路活化的影响。这些结果将阐明油性佐剂免疫增强作用的确切分子机制，促进新的疫苗和药物的研发，也为自身免疫疾病的发病机制研究提供新的线索。

我们前期发现降植烷等油性佐剂可诱导巨噬细胞及自身免疫疾病动物模型自噬增强，但油性佐剂诱导免疫增强的机制仍不清楚。本项目首先采用弗氏佐剂、矿物油、降植烷、鲨烯等油性佐剂刺激巨噬细胞，通过电镜观察、LC3转化检测等反映自噬水平，发现油性佐剂显著诱导细胞自噬增强，同时巨噬细胞炎症分子TLR3及下游细胞因子表达均升高，同自噬的调控相一致；然后，我们通过自噬抑制剂和RNAi干预自噬通路，发现TLR3及下游细胞因子的表达升高受到了抑制，证实自噬介导了油性佐剂触发的TLR3调控等炎症反应；机制方面，我们一方面检测了自噬上游的氧化应激、内质网应激、AMPK和mTOR等通路，发现内质网应激水平升高，mTOR表达受到了抑制，而干预内质网应激则mTOR调节受阻，说明油性佐剂通过诱导细胞内质网应激抑制mTOR通路实现自噬增强；下游方面我们发现STAT1-IRF1信号通路受到活化，参与TLR3的转录调控，而抑制自噬后通路活化受阻；最后我们构建了油性佐剂诱导的自身免疫性关节炎模型，采用慢病毒在体干预自噬，疾病的严重度受到了显著的抑制，从在体水平证实了自噬介导炎症的通路。综上，我们证实了油性佐剂通过诱导细胞自噬增强，活化STAT1信号通路进而调控TLR3，最终实现免疫增强作用。研究结果阐明了油性佐剂免疫增强作用的确切分子机制，将促进新的疫苗和药物的研发，也为自身免疫疾病的发病机制研究提供新的线索。